Advancement of capture immunoassay for real-time monitoring of hepatitis E virus-infected monkey
Graphical abstract
Introduction
HEV infection has become a serious health concern due to the increasing outbreak of Hepatitis E in developing as well as developed countries, which are mostly contributed via waterborne and zootomic-transmitted pathway [1]. Hepatitis E is associated to liver failure and acute liver disease, with higher fatality during pregnancy [2,3]. Recently, major development in HEV detection platform is targeting on the RNA-targeting RT-PCR and antibodies (IgG, IgM and IgA) that were produced after the infection of HEV [4,5]. Due to the limited available diagnostic assay on HEV, a reliable antigen-targeting immunoassay is required to outstand the emerging outbreak by early detection of the disease agent. Instead of RT-qPCR, enzyme-linked immunosorbent assay (ELISA) has been well-known for its simplicity with step-by-step procedure to determine the presence of the target analyte [6,7]. Commonly, a colorimetric-based ELISA utilized peroxidase as the enzyme which catalyzes the oxidation of chromogen substrate by hydrogen peroxide. However, its low sensitivity has been addressed as a strong limitation in detecting HEV type of viral agents, where very low concentration of virus can make the fatality [6]. This issue can be addressed by the signal amplification used in the platform [8,9]. An upgraded ELISA-based biosensor with comparable reliability of RT-qPCR has been in necessity to restrict the outbreak of HEV.
Since the first discovery of Fe3O4 nanoparticles (NPs) exhibiting peroxidase-like activity, known as nanozyme, more application with various kind of nanozymes has been discovered, especially in biosensing technology [[9], [10], [11]]. Various applications such as enzymatic activity [12], nanozymes has been shown to either enhance the activity of the coupled enzymes or exhibiting robust enzymatic-like activity [13,14]. Nanomaterials, especially noble metals, such as gold (Au) [15], silver (Ag) [16] and platinum (Pt) [17], have been employed to substitute the current enzyme used in the immunoassay due to their intrinsic peroxidase-like activity, good stability and biocompatibility [[18], [19], [20], [21]]. Interestingly, hybrid nanomaterial, such as graphene/gold nanohybrid [22], gold-decorated organic framework [23], and bimetallic [[24], [25], [26], [27]] to trimetallic [28,29] nanostructure, demonstrated unique morphology and increasing catalytic activity significantly [30]. To achieve the desired specificity, proper bioconjugation has to be applied successfully. However, in contrast, to maintain the specificity of the nanozyme, bioconjugation can reduce its catalytical activity to some extent [20,31]. To make an optimized condition for modified immunoassays, noble metals have been emerging as a potential platform for easy bioconjugation step with compromising their activity. In this case, Au can be an automatic choice for its less toxicity and cost effectiveness than Pt, and higher plasmonic characteristic compared to Ag in addition with its easy functionalized and control-synthesized properties [32].
In order to develop a highly sensitive nanozyme-based immunoassay for HEV detection, in this study, a signal amplification strategy has been introduced by utilizing silver deposition on Au nanozyme. In our previous study, a proposed immunoassay was demonstrated for norovirus detection and enhanced colorimetric signal in low concentration [20]. Therefore, in this study, more reliable approach was applied to on-site detection and the results were compared to conventional RT-qPCR. However, similar with most of the colorimetric assays, the bare gold nanoparticles (AuNPs) could not be able to perform well in the desired concentration range for real sample analysis. To enhance the performance and stability in real sample medium, in this work, we have proposed a core shell nanocomposite with silver. Silver deposition on gold nanoparticles, was already previously reported for its application in surface-enhanced Raman scattering detection [33], mass spectrometry [34], and opacity densitometry [35], however its applicability is not well explored in the field of catalytic enhancement, especially for virus detection. According to our hypothesis, the AuNPs core with Ag shell structure (AuNPs@Ag) has been introduced for the enhanced catalytic activity of the Ag towards TMB-based color development. The homogeneous distributed AuNPs@Ag with higher stability have been successfully combined to make an upgraded platform for virus detection, especially for real sample analysis. To establish the mechanism, the AuNPs@Ag nanocomposite was synthesized separately and optimized to get the best suited condition for the detection. Then the system was applied in situ deposition during virus-sensing process in the antigen-antibody interaction. A capture immunoassay was demonstrated for HEV detection by using HEV-like particles (HEV-LPs) as the model. By conjugating the Au nanozyme with anti-HEV IgG antibody, the proposed immunoassay demonstrated an amplified signal for low concentration of HEV-LPs. In the presence of silver deposition solution containing silver ions (Ag+) and hydroquinone (HQ) and chromogen solution containing hydrogen peroxide (H2O2) and 3,3′,5,5′-tetramethylbenzidine (TMBZ), the color as a detection signal was significantly amplified compared to the bare AuNP system. In the final step, the nanozyme-based capture immunoassay was challenged to detect the viral HEV containing in fecal samples collected from HEV-infected monkey and showed a comparable trend with RT-qPCR, representing its promising application towards the detection of HEV.
Section snippets
Materials and instruments
Gold (III) chloride trihydrate (HAuCl4·3H2O), bovine serum albumin (BSA), and silver nitrate (AgNO3) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Tetramethylbenzidine (TMBZ) was purchased from Dojindo (Osaka, Japan). Hydrogen peroxide (H2O2), dimethyl sulfoxide (DMSO), hydroquinone (HQ), sulfuric acid (H2SO4), detergent tween-20, and sodium acetate (NaAc) were purchased from Wako Pure Chem., Inc. (Osaka, Japan). Anti-IgG rabbit pAb-conjugated to horseradish peroxidase (HRP) antibody
Principle of silver-shell deposition on gold-core nanozyme
The proposed immunoassay is a modified procedure of capture ELISA assay applying the AuNPs@Ag nanozyme, instead of standard HRP to achieve high sensitivity in rapid process. The process of this proposed immunoassay can be divided in three parts which are schematically presented in Fig. 1 for easy understanding. Initially, the target virus was introduced to the reaction chamber and formed immuno-sandwich structure between immobilized antibodies on the well and Ab-AuNPs via antibody-antigen
Conclusion
The proposed immunoassay utilizing Ag deposition on AuNPs enhanced the peroxidase-like activity significantly as a nanozyme. Silver deposition on the gold showed a significantly amplified signal in the detection of HEV-LPs and HEV compared to conventional HRP-based ELISA as well as the bare AuNP based immunoassays. This proposed immunoassay showed highly sensitive and selective toward the HEV compared to various viral agents even can be observable in naked eyes. The performed assay for HEV-LPs
Ethical approval
The experiments were reviewed by the National Institute of Infectious Diseases (NIID) Ethics Committee and carried out according to the “Guides for Animal Experiments Performed at NIID” under code 514014.
CRediT authorship contribution statement
Indra Memdi Khoris: Writing - original draft, Methodology, Conceptualization. Ankan Dutta Chowdhury: Writing - review & editing, Data curation. Tian-Cheng Li: Resources, Methodology. Tetsuro Suzuki: Data curation, Resources. Enoch Y. Park: Supervision, Writing - review & editing.
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgements
This work was supported and partly by the Bilateral Joint Research Project of the JSPS, Japan. We thank Professor Kouichi Morita of the Institute of Tropical Medicine, Nagasaki University, and Dr. Fuyuki Abe from Shizuoka Institute of Environment and Hygiene, for kindly providing the Zika virus and clinically isolated norovirus. ADC sincerely thank the Japan Society for the Promotion of Science (JSPS) for a postdoctoral fellowship (No. P17359). This research was partially supported by the Japan
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